人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

目的 为了构建人分泌型磷脂酶A2(secretary phospholipase A2, sPLA2-IIA) 的有效表达系统，本文从胎脾中提取总RNA，采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3’末端，构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导，hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达，表达产物以包涵体的形式存在。包涵体经8M尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论：以大肠杆菌为宿主，成功表达了hsPLA2-IIA蛋白，为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。

Gene Coloning, Expression and Enzymatic Assay of Human sPLA2-IIA

Objective To clone the cDNA of human sPLA2-IIA, construct the engineered Escherischia coli expressing human sPLA2-IIA and Identify the expressed human sPLA2-IIA. Methods: Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-IIA was cloned by RT-PCR and inserted into plasmid pET32a(+) between NcoI and EcoRI sites for expressing the recombinant human sPLA2-IIA in Escherischia coli BL21(DE3). The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxA-human sPLA2-IIA fusion protein was established. The expressed human sPLA2-IIA exists in the form of inclusion body and accounts for about 25% of the total soluble proteins of Escherischia coli BL21(DE3). Conclusion: the engineered E. coli methods are suitable for preparing plenty of human sPLA2-IIA which has laid base for the large-scale expression, purification and basic studies of human sPLA2-IIA.